Topic:Beta-Glucuronidase
Beta-glucuronidase is an enzyme that catalyzes the hydrolysis of glucuronic acid from glycosaminoglycans and other glucuronides. In horses, this enzyme is involved in the metabolism of various substances, including hormones, drugs, and toxins. It is present in several tissues and bodily fluids, such as the liver, kidneys, and intestines. The activity of beta-glucuronidase can be indicative of certain physiological and pathological conditions, as it is associated with processes like detoxification and the breakdown of complex carbohydrates. This page compiles peer-reviewed research studies and scholarly articles that explore the role, activity, and implications of beta-glucuronidase in equine health and disease.
Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis. Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacop...
Screening of over 100 drugs in horse urine using automated on-line solid-phase extraction coupled to liquid chromatography-high resolution mass spectrometry for doping control. A fast method for the direct analysis of enzyme-hydrolysed horse urine using an automated on-line solid-phase extraction (SPE) coupled to a liquid-chromatography/high resolution mass spectrometer was developed. Over 100 drugs of diverse drug classes could be simultaneously detected in horse urine at sub to low parts per billion levels. Urine sample was first hydrolysed by β-glucuronidase to release conjugated drugs, followed by centrifugal filtration. The filtrate (1mL) was directly injected into an on-line SPE system consisting of a pre-column filter and a SPE cartridge column for the separa...
In vivo and in vitro metabolism of the designer anabolic steroid furazadrol in thoroughbred racehorses. Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with syntheticall...
Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals. Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and R...
A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry. The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the origina...
Species comparison of oral bioavailability, first-pass metabolism and pharmacokinetics of acetaminophen. Species differences in oral bioavailability, first-pass metabolism and pharmacokinetics of biopharmaceutics classification system (BCS) class I compound acetaminophen were studied. The absolute bioavailability was 42.2%, 39.0%, 44.5%, 75.5% and 91.0% in chickens, turkeys, dogs, pigs and horses, respectively. After hydrolysis of metabolites by beta-glucuronidase/sulfatase, apparent bioavailability increased significantly in all species (turkeys: 72.4%, dogs: 100.5%, pigs: 102.2%), except horses (91.6%). Mean metabolic ratios of [acetaminophen glucuronide]/[acetaminophen] between 0 and 1h were s...
Alkaline and acid phosphatase, β-glucuronidase and electrolyte levels in fractionated stallion ejaculates. Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β-glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections we...
Pyrilamine and O-desmethylpyrilamine detection in equine serum and urine. Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days ...
Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry. A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-...
Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry. The metabolism of clemastine was studied in dogs, horses, and humans after a single dose of Tavegyl. The urine collected was extracted by solid-phase extraction or hydrolyzed with beta-glucuronidase and then extracted by liquid-liquid extraction, prior to analysis for unchanged drug and phase I and II metabolites by liquid chromatography/tandem mass spectrometry. The metabolites were identified by their molecular mass and interpretation of the product ion spectra, since no standard substances were available. Unchanged drug was recovered in urine samples from dogs and humans, but not from horse...
Pharmacodynamics and enantioselective pharmacokinetics of racemic carprofen in the horse. Carprofen is a nonsteroidal anti-inflammatory drug of the 2-arylpropionate subclass. It contains a single chiral centre and exists in two enantiomeric forms. In this study rac-carprofen, at two dosages, 0.7 and 4.0 mg/kg, and placebo were administered i.v. to six New Forest horses in a three period cross-over study. The concentration-time profiles were established for R(-) and S(+)-carprofen for plasma and both inflamed (exudate) and noninflamed (transudate) tissue cage fluids. R(-)-carprofen was the predominant enantiomer in all three fluids, as indicated by plasma area under the curve (AUC) ...
Proteins and enzymes in uterine lavage fluid of postpartum and nonparturient mares. Uterine lavage fluids from postpartum and nonparturient mares were compared to determine when the normal secretory capacity of the postpartum uterus is restored. Lavage fluids were obtained from cyclic nonparturient mares on the second, fourth or fifth day of oestrus, and 3, 8, or 14 days after ovulation (seven mares/sampling day). Twelve intact postpartum mares were sampled 1 to 28 days postpartum (group A: 1, 6, 12 and 20; group B: 2, 8, 14 and 24; group C: 4, 10, 16 and 28 days postpartum; four mares/group). Three ovariectomized (OVX) postpartum mares were sampled as mares in group C. Sampl...
Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares. The aim of the study was to determine whether neutrophil numbers (PMN), trypsin-inhibitor capacity (TIC), lysozyme, N-acetyl-beta-D-glucosaminidase (NAGase), beta-glucuronidase (B-Gase), total protein, and plasmin in uterine lavage fluid of postpartum (p.p.) mares, either at the time of foal heat insemination or around the time of arrival of the embryo in the uterus, could be used in predicting conception. Fifteen mares were inseminated within 13 h after the first p.p. ovulation. Uterine lavage fluids were successfully collected from 9 out of 12 mares before insemination and from all 15 mares ...
Identification of hydroxyropivacaine glucuronide in equine urine by ESI+/MS/MS. Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following beta-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI+/MS/MS, the presence of a [M+H]+ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide +1. The abundance ...
Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine. Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight hor...
Gelatinolytic activity in tracheal aspirates of horses with chronic obstructive pulmonary disease. The gelatinolytic activity in tracheal aspirates (TA) of horses with chronic obstructive pulmonary disease (COPD) was analyzed using SDS-PAGE-gelatin-gel electrophoresis (zymography) and compared to TAs from healthy controls. The 110-90 kD MMP-9 type gelatinase was high in symptomatic disease phases (permanent disease 0.46 +/- 0.15, p < 0.001; or intermittent disease 0.47 +/- 0.12, p < 0.001) compared to healthy controls (0.10 +/- 0.07). Similarly, the overall gelatinolytic activity, the activity in high-mw gelatinolytic bands (210-190 and 150 kD) and in proteolytically processed fragments in ...
Pharmacokinetics and pharmacodynamics of ketoprofen enantiomers in the horse. Pharmacokinetic and pharmacodynamic parameters were established for enantiomers of the non-steroidal anti-inflammatory drug (NSAID) ketoprofen (KTP), each administered separately at a dose level of 1.1 mg/kg to a group of six New Forest geldings, in a three-period cross-over study using a tissue cage model of inflammation. For both S(+)-and R(-)-KTP, penetration into tissue cage fluid (transudate) and inflamed tissue cage fluid (exudate) was rapid, and clearances from exudate and transudate were much slower than from plasma. AUC values were, therefore, higher for exudate and, to a lesser degre...
Effects of flunixin, tolfenamic acid, R(-) and S(+) ketoprofen on the response of equine synoviocytes to lipopolysaccharide stimulation. The objective of this study was to analyse the effects of 4 nonsteroidal anti-inflammatory drugs (NSAIDs) on the production of beta-glucuronidase (beta-glu), tumour necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated equine synoviocytes. The agents studied were flunixin, tolfenamic acid, S(+)ketoprofen (KTP) and R(-)ketoprofen. LPS-induced release of beta-glu from synoviocytes was inhibited in a concentration dependent manner by all 4 compounds, tolfenamic acid being the most potent. Of the 2 KTP enant...
Determination of phase II drug metabolites in equine urine by micellar electrokinetic capillary chromatography. Micellar electrokinetic capillary chromatography (MECC) using diode array detection has been investigated for the determination of phase I and phase II metabolites of drugs in biofluids. Methods were optimised for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide, normorphine, meclofenamic acid and its metabolites in equine urine. Solid-phase extraction procedure were developed to concentrate and purify the analytes from spiked and post administration urines for MECC analysis. A simple on-line procedure for monitoring the kinetics of hydrolysis of morphine-glucuroni...
Exercise-induced changes in the activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase in plasma and muscle of standardbred trotters. The activities of lysosomal enzymes, such as beta-glucuronidase and N-acetyl-beta-D-glucosaminidase, have been shown to increase in muscle after endurance exercise. We examined whether measurable activities of lysosomal enzymes are present in equine plasma and whether the exercise-induced changes in the muscle are reflected in plasma. Six trained Standardbred trotters performed three exercise bouts with 1 h intervals and the same procedure was repeated 3 days later. Venous blood samples and muscle biopsies from the middle gluteal muscle were taken before and after exercise. The activities of b...
Identification of metabolites of azaperone in horse urine. Two metabolites of the tranquilizer azaperone were extracted from alkalinized horse urine after treatment with beta-glucuronidase/sulfatase from limpets (Patella vulgata). The metabolites were identified by a combination of independent chemical synthesis and GC/MS and 1H NMR analysis. The metabolites were identified as 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanol, designated as 5'-hydroxy-azaperol, and 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanone, designated as 5'-hydroxyazaperone. A TLC screening test was developed for detecting both metabo...
Postmortem tissue samples: an alternative to urine and blood for drug analysis in racehorses. Although urine is the sample of choice for drug tests in racehorses, it is rarely obtained following the sudden death of a racehorse on the track while racing. The purpose of this study was to demonstrate the significance of postmortem tissue samples as an alternative to urine and blood samples in equine drug analysis following the sudden death of a racehorse on the track while participating in a competitive race. Postmortem tissue samples were frozen (-80 degrees C) until analyzed. A 30-40-g portion of each organ was homogenized in a 0.1 M phosphate buffer (pH 7.4), deproteinized, hydrolyzed ...
Beta-glucuronidase and trypsin inhibitor capacity of tracheal lavage fluid as indicators of seasonal airway irritation in the horse. The health of the respiratory tracts of 19 horses was studied for 11 months. The horses were placed into 3 groups (healthy, periodically diseased and continuously diseased) based on the measurements of blood gases, intrapleural pressure and on neutrophil content of tracheal mucus. Lysosomal enzymes (N-acetyl-beta-D-glucosaminidase and beta-glucuronidase) and reflectors of the proteolytic system (plasmin, plasminogen, trypsin inhibitor capacity) were determined. beta-glucuronidase appeared to be a good indicator of the presence of disease of the respiratory system. High beta-glucuronidase value...
Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide. Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than ...
Effects of allopurinol in experimental endotoxin shock in horses. The effect of allopurinol pretreatment 12 hours before an intraperitoneal challenge with a sublethal dose of Escherichia coli endotoxin (50 micrograms kg-1) was evaluated in 18 horses. The horses were divided among three equal groups: 1-endotoxin alone; 2-5 mg allopurinol kg-1 bodyweight plus endotoxin; and 3-50 mg allopurinol kg-1 bodyweight plus endotoxin. A variety of evaluation parameters were used. No differences among the groups were noted in rectal temperature, heart rate, respiration rate, haematological values, blood PaO2, blood PaCO2, blood pH or blood bicarbonate. Significant (P les...
Metabolism and pharmacokinetic studies of propionylpromazine in horses. The propionylpromazine concentrations in plasma after intramuscular administration to horses were determined using gas chromatography with nitrogen-phosphorus detection. After hydrolysis by beta-glucuronidase/arylsulphatase, the parent drug and three metabolites were detected in urine. The metabolites were identified as 2-(1-hydroxypropyl)promazine, 2-(1-propenyl)promazine and 7-hydroxypropionylpromazine by gas chromatography-mass spectrometry. No N-demethylated or sulphoxidated metabolites of propionylpromazine were observed in the horse urine.
Effects of nandrolone phenylpropionate in the horse: (3) skeletal muscle composition in the exercising animal. The effect of 11 weekly injections of nandrolone phenylpropionate (400 mg) on some skeletal muscle parameters was investigated in 6 Thoroughbred geldings undergoing training. Three muscles were sampled, the middle gluteal, the biceps femoris and the semitendinosus. Training alone produced increases in the percentage of fast twitch high oxidative fibres (FTH), glycogen content and the activities of citrate synthase, 3-hydroxyacl CoA dehydrogenase and cytochrome oxidase. In contrast the training programme did not alter water content, total protein content, the activities of lactate dehydrogenase...